Test for thyroid hormone

ABSTRACT

A procedure utilized in a diagnostic test for determining the level of thyroid hormone within a body fluid which includes admixing a radioactive isotope labeled hormone with a body fluid, and separating the resulting unbound hormone from the hormone bound by the hormone binding globulin and protein of the fluid by sorption of the unbound hormone by a particulate inorganic crystalline material. This sorption does not depend upon critical time or temperature limitations, and is generally conducted by thoroughly admixing the sorbent material with the solution containing the bound and unbound thyroid hormone at room temperature. The sorbent material is then separated from the resultant supernatant fluid and either the supernatant fluid or sorbent material, preferably the sorbent material, is counted with a scintillation counter to determine the labeled hormone bound to or free from the globulin.

Unite States Patent Eisentraut [72] Inventor:

[73] Assignee: Nuclear-Medical Laboratories, Inc., Dallas, Tex.

[22] Filed: July 30, 1969 21 Appl. No.: 846,289

[52] U.S.Cl ..424/l,250/106 T, 424/111, 424/357 [51] Int. Cl. ..A61k27/04 [58] Field of Search ..424/1, 111, 357; 23/230; 250/106 T [56]References Cited UNITED STATES PATENTS 3,414,383 12/1968 Murphy ..23/2303,451,777 6/1969 Di Guilio .424/1 X 3,516,794 6/1970 Murty et al. 424/1X Primary ExaminerBenjamin R. Padgett Att0rneyRichards, Harris & Hubbard[5 7] ABSTRACT A procedure utilized in a diagnostic test for determiningthe level'of thyroid hormone within a body fluid which includes admixinga radioactive isotope labeled hormone with a body fluid, and separatingthe resulting unbound hormone from the hormone bound by the hormonebinding globulin and protein of the fluid by sorption of the unboundhormone by a particulate inorganic crystalline material. This sorptiondoes not depend upon critical time or temperature limitations, and isgenerally conducted by thoroughly admixing the sorbent material with thesolution containing the bound and unbound thyroid hormone at roomtemperature. The sorbent material is then separated from the resultantsupernatant fluid and either the supernatant fluid or sorbent material,preferably the sorbent material, is counted with a scintillation counterto determine the labeled hormone bound to or free from the globulin.

9 Claims, No Drawings TEST FOR THYROID HORMONE This invention relates todiagnostic tests for the determination of the level of thyroid hormonewithin a body fluid. In another aspect, this invention relates todiagnostic tests which measure unsaturated binding capacity of hormonebinding globulin and other proteins, and measure total thyroid hormonein blood serum. In still another aspect, this invention relates to animproved method for removing free thyroid hormones from mixturescontaining hormones in a free state and hormones which are bound tohormone binding globulin and other proteins.

Various diagnostic tests are known in the art for determining thyroidfunction. These tests include the basal metabolism test, the thyroiduptake test and various colorimetric and chemical procedures fordetermining the level of thyroxine iodine in the blood. Among the mostaccurate tests available are the diagnostic tests which utilizeradioisotope labeled hormone to indirectly determine the level ofthyroid hormones, thyroxine (C, I-I,,I,,NO,,), and triiodothyronine (C,H, I NO,,) present in body fluids. Specifically, these tests include atest commonly referred to as the T-3 test which measures the unsaturatedbinding capacity of thyrobinding globulin and other proteins within abody fluid such as blood, and the test commonly referred to as the T4test which measures the total quantity of hormone within a sample ofblood serum.

Both of these tests include the steps of adding the radioisotope labeledhormone to a solution containing a sample of hormone produced within thebody and thyrobinding globulin, separating the resulting thyrobindingglobulin containing bound hormone from the resulting unbound hormone,and counting the radioactivity of either the bound or unbound hormone.This counting procedure will indirectly indicate the amount ofendogenous hormone which is bound to the natural globulin and proteinbounding sites within the blood.

Thus, both the T-3 and the T4 tests depend for their accuracy upon theefficient separation between the bound and unbound thyroid hormone inthe test sample. The currently available methods for removal of thesehormones include ion exchange resins such as the ion exchanger havingstrongly basic amino or quatenary ammonium groups as disclosed in U.S.Pat. No. 3,414,383. These organic ion exchange resins can be either inloose forms, or incorporated in polyurethane sponges as disclosed inU.S. Pat. No. 3,206,602, or enclosed in porous bags or the like. Anotheravailable method involves a selective adsorption of the free hormones bycharcoal which has been coated with suitable proteins.

The major disadvantages of all the currently available separationmethods described above is that the binding of the hormones to theseparation materials is both time and temperature dependent, so that thebinding of the hormones to these materials increases with time andincreasing temperatures. For example, as time increases, much of theproteinbound thyroid hormone is ultimately bound to the ion exchangeresin. In addition, from one to two hours are required for completebinding to take place with the ion exchange resins, further limiting thenumber of measurements which are possible in the laboratory. Also,coated charcoal must be enclosed within a heat block under controlledtime and temperature conditions during the separation procedure. Thus,careful timing and controlled temperatures 5 are necessary in theconventional T-3 and T-4 test when separating the thyroid hormones boundto the natural binding sites within the blood (thyrobinding globulin andother proteins) from the nonbound thyroid hormones.

Therefore, one object of this invention is to provide improveddiagnostic tests for determining the level of thyroid hormone in a bodyfluid.

Another object of this invention is to provide an improved method ofseparating thyroid hormone bound to thyroid-binding globulin and otherproteins from nonbound thyroid hormones which method is not criticallydependent on time or temperature conditions.

According to the invention, the separation of free hormones fromhormones bound to natural binding sites (thyrobinding globulin and otherproteins) in a diagnostic test is carried out by sorption of the freehormones by particulate inorganic crystalline lattice material,preferably in colloidal form. It has been discovered that the sorptionof the free hormones by the inorganic crystalline material is neithercritically time nor temperature dependent, and results in highlyefficient, fast, and reproducible results. This separation is conductedby adding a measured amount of the inorganic crystalline sorptionmaterial to the sample, thoroughly admixing the sorption material withthe solution for a few seconds, and separating, (such as centrifugation)the resultant supernatant fluid containing the protein-bound hormonesfrom the resulting material containing the free hormones. Either thesupernatant fluid or the colloidal material can be counted in ascintillation well counter; however, it is preferred that the colloidalmaterial be counted.

The particulate inorganic crystalline material which can be used withinthe scope of this invention includes the phosphates, oxides, hydroxides,silicates, aluminates, and sulfates, of the metallic elements in groupsIA, IIA, llIA, HE and VIII of the Periodic Table as illustrated on pageB-2 of the Handbook of Chemistry and Physics, Chemical Rubber PublishingCompany (1964). Examples of suitable materials include calciumcarbonate, calcium phosphate, calcium oxide, calcium hydroxide, calciumsilicate, calcium aluminate, calcium sulfate, magnesium carbonate,magnesium phosphate, magnesium oxide, magnesium hydroxide, magnesiumsilicate, magnesium aluminate, magnesium sulfate, aluminum carbonate,aluminum phosphate, aluminum oxide, aluminum hydroxide, aluminumsilicate, aluminum sulfate, potassium carbonate, potassium phosphate,potassium oxide, potassium hydroxide, potassium silicate, potassiumaluminate, potassium sulfate, iron carbonate, iron phosphate, ironoxide, iron hydroxide, iron silicate, iron aluminate, iron sulfate,barium carbonate, barium phosphate, barium oxide, barium hydroxide,baRium silicate, barium aluminate, barium sulfate, zinc carbonate, zincphosphate, zinc oxide, zinc hydroxide, zinc silicate, zinc aluminate,zinc sulfate, and mixed salts thereof.

Some specific examples of commonly occurring materials which can be usedwithin the scope of this invention include: Opal, (Si(OH),+SiOWaterglass, Si,,O Na Kaolinite, Al.,,(SiO ,oh.,,; Dickite, Al (Si O)(OI-l),; Nacrite, Al (Si O )(OH),; Metahalloysite, Al (Si O )(OH),,;Halloysite, Al (SiO,,,oh. Attapulgite, Mg;,(Si.,O )(Ol-I) (OI-I)-2l-I O,Al(Si.,O,,,)(OI-I) Pyrophyllite, Al (Si,O, )(OI-I) Ialc,

The most preferred inorganic crystalline sorbent materials include thesilicates, particularly magnesium silicate and aluminum silicate. Otherpreferred sorbent materials are calcium phosphate, silicic acid,aluminum hydroxide, calcium oxide, and magnesium oxide.

The inorganic crystalline sorbent material is preferably in colloidalform (an average diameter of from 10" to 10 centimeters). However, thesorbent material can have a non-colloidal particle size of from 4 meshto 325 mesh. (U.S. Standard). When using non-colloidal particles, it ispreferred that particle size range from 10 mesh to 325 mesh (U.S.Standard).

In operation the particulate inorganic sorbent material is added to thetest sample and thoroughly admixed therewith. For example, a preweighedamount of sorbent is added to the test sample within a test tube, andthoroughly admixed for example, for about 30 seconds, by touching thebottom of the test tube to a Vortex mixer. After a period of 2 minutes,the test tube can be placed within a conventional centrifuge to separatethe inorganic sorbent material from the resulting supematant fluid. Thisprocedure can occur at any convenient temperature, for example, roomtemperature. Additionally, the period of contact between the inorganicsorbent material and the test sample is not critical. For example, thematerial can be allowed to sit for any convenient period of time afterthe mixing. It is noted that the sorbent should remain in contact withthe test sample for a period of at least about 2 minutes before thecentrifugation step.

This invention basically constitutes improved diagnostic tests whichmeasure either of l) unsaturated binding capacity of the thyrobindingglobulin and other proteins within body fluids, or (2) the total serumthyroxine within body fluids. The body fluid being treated is normallyserum, but unknown amounts of thyroid hormone (thyroxine andtriiodothyronine) can be determined in blood plasma or whole blood, andthe like. The basic improved tests of this invention are described asfollows:

TEST FOR UNSATURATED BINDING CAPACITY OF THYROBINDING GLOBULIN AND OTHERPROTEINS In this test, a tracer quantity of radioactive isotope labeledhormone is admixed with a known amount of body fluid (serum), theinorganic sorbent material is added to the resultant sample solution,thoroughly admixed therewith, and then separated therefrom such as bycentrifugation. At this point, either the resulting supernatant fluidfrom the test solution, or the separated inorganic sorbent material canbe counted in a scintillation well counter to determine the amount ofradioactive isotope labeled hormone therein. This determination willindirectly show the level of hormone which was originally bound to thebinding sites, thyrobinding globulin and other proteins within theserum. A preferred method is as follows:

Initially a tracer quantity of radioactive isotope labeledtriiodothyronine or thyroxine, preferably triidodothyronine, is dilutedin 0.1 percent albumin in barbital buffer (diethyl bar bituric acid pH8.6,0.075 M). The hormone utilized is labeled with either radioactive Ior I (or theoretically, any radioactive isotope of iodine, tritium,nitrogen or carbon). The P is preferably because it has a half life of60 days and it can be employed for at least 6 weeks; whereas, I has ahalf life of 8 days and has a useful shelf life of about 2 weeks. Theresulting mixture is added to two milliliters of barbital buffer (pH8.6,0.075 M). It is noted that the barbital buffer could be employedwith an alternate pH and molarity and other buffers could be used. Forexample, the barbital buffer can be used in a pH range of from 6.8 to9.6, and other buffers such as tris (hydroxymethyl) aminomethane, tris(hydroxymethyl) aminomethane-maleate, sodium phosphate, and potassiumphosphate can be used. It is possible with these other buffers tooperate at a pH as low as about In addition, saline solution ordistilled water can be used in place of the buffer.

Next, 0.1 milliliter of the sample serum (from the patient) is added tothe resulting mixture. It is noted that various volumes of the serum canbe employed, but this amount is preferred in that it yields the greatestdifference between normal and abnormal sera.

At any convenient time after the serum is added, the particulateinorganic crystalline sorbent can be added. For example, pre-weighedaliquots of any of the above described inorganic crystalline sorbentscan be utilized. Other quantities of these sorbents may be employed forexample, from 20-600 mg, or more. however, optimum quantities wereselected as follows. A tablet in which 50 milligrams of Mg -,(SiO,,O)(OH is bonded with 80 milligrams of acacia is added to each samplewithin a test tube. Suitable tablets are produced by GoldleafPharmaceutical Company, New Jersey. After the addition of the tablet tothe test tube, the sample is vigorously agitated for about 30 seconds bytouching the bottom of the test tube to a Vortex mixer.

At any convenient time, for example, between 5 minutes and 2 hours afterthe cooloidal material is suspended within the sample solution, the testtubes are centrifuged to separate the colloid from the resultingsolution. For example, the material is centrifuged at approximately2,400 rpm for 5 minutes. The resulting supernatant fluid is then pouredoff, and the test tube turned upside down to drain on a gauze sponge.The colloidal material is packed very tightly in the bottom of the tubeand is not removed by the latter drainage procedure.

At this point, either the discarded supernatant fluid or the packedsorbent can be counted in the scintillation well counter. However, it ispreferred that the packed sorbent be counted. The per cent freeradioactive hormone is determined by determining the ratio of the samplecounts per minute from the solvent material to the total counts perminute of standard amount of radioactive hormone which is equivalent tothe total radioactive hormone initially added to the sample. This isaccomplished by preparing a standard sample of the radioactive hormonediluted with water to the same approximate volume as the packed sorbentoccupies in the bottom of the test tube (about 0.2 milliliters), andcounting the resulting solution to establish the standard counts perminute. In this manner the counting efficiency of this standard will beidentical to the counting efficiency of the unknown.

The normal range employing the above described method with 50 milligramsof the magnesium silicate is 34-44 percent. Hypothyroidism and pregnancyyield values below the normal range while values in hyperthyroidpatients are higher.

TEST FOR TOTAL SERUM THYROXINE Basically, this test is directed towardthe measurement of the total amount of thyroxine within a sample ofserum by competitive binding" to thyrobinding globulin, wherein thethyroxine is initially liberated from serum proteins, and ad mixed witha standard solution of thyrobinding globulin and labeled thyroxine.After this, the unbound thyroxine is removed from the resulting solutionwith the inorganic crystalline sorbent and then separated from theremaining labeled thyroxine which is bound to the globulin, Either thebound or the free fraction is then determined by counting techniques (ina scintillation well counter).

The thyroxine is liberated from serum proteins by either precipitationor denaturation. This can be accomplished with a variety of organicsolvents such as methanol, ethanol, ethanol and n-butanol, acidified 2,2 dimethyoxypropane, and the like. Since, regardless of the solventemployed, some of the endogenous thyroxine will be lost in the proteinprecipitate, it is preferable to obtain individual recovery values oneach serum sample assay. This is true because the use of so-calledaverage recovery value proposed by most investigators results in anerror of approximately 1 9 percent. A preferred method of determiningpercent recovery is as fol lows: A solution (hereinafter called SolutionA) containing approximately 50 microcuries of thyroxine containingeither I or I l5 milliliters of normal human serum and 5 milliliters ofpropylene glycol is diluted in 500 milliliters of barbital buffer (pH8.6,0.075 M). Next, 0.1 milliliter of this solution is added to each 1milliliter serum sample. At this time, in order to calculate individualrecovery values, 0.1 milliliter of this solution is also added to 0.4milliliter of water and set aside for derivation of total counts perminute. To each of these one milliliter serum samples, 4 milliliters of5 percent methanol in percent ethanol is then added thereto within atest tube. The resulting solution is mixed on a Vortex mixer for about30 seconds and is allowed to set for about 5 minutes thereafter. Thetest tubes are then centrifuged at speeds to yield a completely clearsupernatant. The protein precipitate is discarded. Next, 0.5 milliliterof the supernatant fluid is counted and compared with the counts derivedfrom the standard sample to determine the percent recovery of thyroxinefrom each sample.

After this, 0.5 milliliters of the supernatant from each sample testtube is transferred to another test tube and dried under forced air ornitrogen. Drying may be expedited by placing the tubes in a water bathmaintained at 45-60 C. The drying operation will take approximately 45minutes. The drying operation will effectively remove the solventtherefrom and leave a deposit of thyroxine in the tube. After the dryingoperation, one milliliter of the radiosotope labeled thyroxine solutiondescribed above (Solution A) is added to each sample test tube. Themixture is then shaken to assure the complete dissolution of thepreviously dried material contained therein. For example, each tube isshaken three times during a minute equilibration period.

After the dissolution of the dried precipitate within the radioactiveisotope solution, 50 milligrams of the inorganic crystalline sorbent isadded to remove the free thyroxine therefrom, and the percent ofradioactivity sorbed to the colloid is determined. If desired, thepercent radioactivity within the resulting supernatant can bedetermined; however, it is preferred to determine the percent ofradioactivity contained within the sorbent.

The microgram concentration of thyroxine is next determined from astandard curve. The standard curve can be prepared as follows:

Unlabeled thyroxine (sodium levothyroxine which is equivalent to 0.05micrograms of thyroxine per milliliter) is diluted in 0.1 percentalbumin diluted in a barbital buffer. From this solution, four standardsolutions are prepared. The standard solutions contain 5, 10, I5 and 20nanograms, respectively, of the unlabeled thyroxine as prepared above inb l milliliter of the radioisotope labeled thyroxine standard solution(Solution A). An additional milliliter of this standard solution isprepared with no unlabeled thyroxine added. 50 milligrams of theparticulate inorganic crystalline material, e.g., colloidal magnesiumsilicate, are added to each of the above-described 5 standard solutionsto determine the percent of free thyroxine in the manner as describedpreviously, After this determination, a standard curve is plotted onlinear graph paper with nanogram or microgram concentration (rangingfrom O to 20 nanograms) plotted on the abscissa, and percent thyroxinebound to the sorbent plotted on the ordinate.

lt is noted at this point since this assay utilizes the so-calledprinciple of competitive protein binding, an increase in the totalhormone concentration decreases the percentage of labeled hormone whichcan be bound to a fixed quantity of standard serum. Thus, since thecolloid binds only the free thyroxine, the percent of radioactivitywhich is counted will increase as the concentration of thyroxine iselevated. Thus, it is preferred that concentration of the standard serumand the amount of the particulate inorganic crystalline sorbent employedshould be adjusted to yield a percent binding (by the sorbent) whichranges from about 20 to about 60 percent. After the standard curve hasbeen formed, the thyroxine concentration of each unknown test sample isobtained therefrom and corrected for dilution and percent recovery.Thus, in this example:

aliquot concentration X 10.2

These values are usually corrected to be reported as micrograms per 100milliliters (microgram percent) of serum. The normal range is 5.2 to13.8 microgram percent. Thus, since thyroxine is 65.3 percent iodine,the normal range will be equivalent to 3.4-9 microgram percent ofthyroxine iodine.

lt is noted that in the separation procedure of this test, and in theunsaturated binding capacity test, only a 2 minute incu bation period isrequired for the sorbent to completely bind all of the unbound hormones,yet prolonged incubation does not result in sorption of protein boundhormone. In addition, the sorption proceeds at normal room temperaturesmaking previously required temperature control mechanisms, e.g., icebaths and/or heaters, unnecessary. It is generally preferred that thesorption process of this invention be carried out at a temperaturewithin the range of from about 20 to about C. (68 86 F).

lt is within the scope of this invention to provide the basicingredients of both of the above described diagnostic tests in packagedform. For example, the test kit can comprise packaged units, e. g., fromabout 20 to about 600 milligrams or more of the inorganic crystallinesorbent material. The sorbent material can be present in a compressedtablet form such as for example, magnesium silicate or silicic acid orany of the other described materials combined with a suitable binder,such as acacia gum or the like. The packaged kits can comprise thesorbent, a barbital buffer solution sufiicient to pro vide an optimalpH, and a radioisotope labeled thyroxine or triiodothyronine solution.In addition, the kit can contain a standard thyrobinding globulinesolution.

This invention can be more easily understood from a study of thefollowing examples which are given for illustrative purposes only.

EXAIVIPLE 1 In this Example, the unsaturated binding capacity test wasconducted utilizing the technique described above wherein theradioisotope labeled hormone was triidothyronine (labeled with 1 Tensamples were prepared using the 0.1 milliliter of serum as described inthe above test.

The tests were conducted according to the procedure outlined abovethrough the point at which the tablet containing 50 milligrams ofmagnesium silicate was added to the sample solution and thoroughlyadmixed therewith. However, at this point, the various solutions werecentrifuged and then counted in a scintillation well counter aftervarious time periods. The first sample was centrifuged immediately, andthe results showed 29.9 percent of the hormone was removed by thesorbent. After two minutes the second sample was centrifuged and showedthat 34.6 percent of the hormone was removed by the sorbent. Next, thethird, fourth, fifth, sixth, seventh, eighth and ninth samples werecentrifuged after 5, l0, 15, 30, 45, 60 and minutes, respectively. Thecounting evidenced no deviation in percent of the hormone bound, andtherefore no increase in uptake of the hormone by the sorbent material.The tenth sample was centrifuged and counted after minutes to show that34.9 percent of the hormone was removed by the sorbent.

The above tests clearly indicate that the time period is not critical inwhich the inorganic sorbent material must function. This sorbentmaterial substantially immediately binds all the free hormones withinthe test samples, and will not either release sorbed hormone or continueto sorb hormone bound to the thyrobinding globulin or other proteins inthe sample as time progresses. This quality is a definite advantage overthe use of the ionic exchange resins and the coated charcoal of theprior art, since these materials can only be placed in contact with thesample solution for controlled periods of time in order to obtainreproducible results. In addition, the inorganic crystalline absorbentmaterial of this invention functions uniformly and efiiciently in normalroom temperature conditions; whereas, the prior art material must beutilized under controlled temperature conditions.

EXAMPLE 2 Next, the unsaturated binding capacity test as describedabove, and using triidothyronine labeled with radioactive I wasconducted simultaneously on 10 samples of the same serum. The 10 testswere conducted simultaneously according to the exact procedure outlinedabove. The resulting percent hormone bound (to the sorbent) for the 10samples were as follows: 33.6, 33.4, 33.3, 33.4, 33.!1, 33.3, 32.9,31.5, 33.7, 34.7. The results indicated a standard deviation of only0.79 and illustrate that the use of the inorganic crystalline sorbentmaterial yields highly reproducible results.

EXAMPLE 3 The basic unsaturated binding capacity test which was utilizedin the two above Examples was conducted on control normal serum,hypothyroid serum, and hyperthyroid serum except the quantity of serumand magnesium silicate, and total solution was varied in each run.

RUN I In this run, 0.2 milliliters each of the control sera was utilizedtogether with 25 milligrams of magnesium silicate, and a total barbitalsolution volume of 2 milliliters. The results of the runs were asfollows (percent hormone taken up by sorbent):

hyperthyroid serum 22% normal serum 13% hypothyroid serum 8.6%

RUN 2 In run 2, 0.2 milliliters each of the control sera was utilizedtogether with 50 milligrams of magnesium silicate and a total solutionvolume of 2 milliliters. The results were as follows (percent hormonetaken up by sorbent):

hyperthyroid serum 41.5% normal serum 23.5% hypothyroid serum 16% RUN 3In this run 0.2 milliters each of the control sera was utilized togetherwith 100 milligrams of magnesium silicate and a total solution volume of2 milliliters. The results were as follows (percent hormone taken up bythe sorbent):

hyperthyroid serum 56.5% normal serum 42.1% hypothyroid serum 27% RUN 4in this run, 0.1 milliliters each of the control sera was used togetherwith 50 milligrams of magnesium silicate and a total sample solutionvolume of 2 milliliters. The results were as follows (percent hormonetaken up by the sorbent):

hyperthyroid serum 54.8% normal serum 34.6% hypothyroid serum 23% RUN 5in this run 0.1 milliliter each of the control sera was utilizedtogether with 50 milligrams magnesium sulfate and 3 milliliters totalsample solution. The results were as follows: (percent hormone taken upby sorbent):

hyperthyroid serum 55.8% normal serum 33.5% hypothyroid serum 21.5%

RUN6

in this run 0.] milliters each of the control sera was utilized with 100milligrams magnesium silicate and 3 milliliters total sample solution.The results were as follows (percent hormone taken up by sorbent):

hyperthyroid serum 69% normal serum 48.5% hypothyroid serum 38%separations. The combinations utilized in Run 4 has been foundpreferably because it will conveniently provide broad separationsbetween hypothyroid, normal and hyperthyroid while utilizing a minimumquantity of sample materials.

While this invention has been explained in relation to its preferredembodiments, it is to be understood that various modifications thereofwill now become apparent to one skilled in the art upon reading thespecification, and it is intended to cover such modifications as fallwithin the scope of the appended claims.

I claim:

1. In a test for thyroid hormone wherein a known quantity of radioactiveisotope labeled thyroid hormone is admixed with a sample fluidcontaining an unknown quantity of said thyroid hormone, and thyroidhormone binding globulin to determine the amount of said unknown hormoneby counting with a scintillation counter one of (a) said labeled hormoneboundto said hormone binding globulin, and (b) said labeled hormonewhich is not bound to said thyroid hormone binding globulin as theresult of said admixing, the improvement comprising:

thoroughly admixing said sample fluid containing said known quantity ofradioactive isotope labeled hormone with a particulate inorganiccrystalline sorbent material selected from silicic acid and carbonates,phosphates, oxides, hydroxides, silicates, aluminates, and sulfates ofthe metallic elements in Groups lA, llA, lllA, HE, and VIII of thePeriodic Table, and mixed salts thereof for a sufficient time to causethe selective sorption to said sorbent material of hormone which is notbound to said thyroid hormone binding globulin, and separating saidsorbent material from the resulting sample fluid before said counting.

2. The improved test of claim 1 wherein the said particulate inorganiccrystalline sorbent material comprises colloidal magnesium silicate.

3. The improved test of claim 2 wherein said sorbing occurs at atemperature within the range of from about 20 C to about 30 C.

4. The method of claim 3 wherein said sorbing occurs during a timeinterval of at least about 2 minutes.

5. The improved test of claim 4 wherein said sample fluid comprisesblood.

6. A method for determining the level of thyroidhormone in a sample ofbody fluid containing an unknown quantity of said hormone and hormonebinding globulin comprising:

a. thoroughly admixing a solution of this sample with a solutioncontaining a radioactive isotope labeled hormone and barbital andallowing the resulting solution to equilibrate;

b. thoroughly admixing the resulting equilibrated solution from step (a)with a particulate inorganic crystalline sorbent material selected fromsilicic acid and carbonates, phosphates, oxides, hydroxides, silicates,aluminates, and sulfates of the metallic elements in Groups IA, llA,"IA, 118, and VIII of the Periodic Table, and mixed salts thereof toremove free thyroid hormone from said solution by selective sorptionthereof by said sorbent materic. separating said sorbent from saidsolution; and

d. counting with a scintillation counter one of l) the free radioisotopelabeled hormone in said sorbent, and (2) the bound radioisotope labeledhormone in said resulting solution.

7. The method of claim 7 wherein said sorbent material is colloidalmagnesium silicate.

8. A method of measuring thyroxine in a sample solution comprising:

a. precipitating proteins from said sample solution by admixing saidsample solution with an organic solvent and thereby leaving saidthyroxine in solution;

b. thoroughly admixing a solution of the thyroxine from step (a) with asolution containing thyrobinding globulin,

9 10 radioactive isotope labeled thyroxine, and barbital and alsaidsorbent material; lowing the resulting solution to equilibrate; d.separating said sorbent material from the resulting solu- C. thoroughlyadmixing the equilibrated solution of step (b) tion; and

with a particulate inorganic crystalline sorbent material C. countingwith a scintillation counter n Of (1) free selected from silicic acidand carbonates, phosphates, oxlabeled thyrwfine l y in Said and ides,hydroxides, silicates, aluminates, and sulfates of the labeled thyroxmefiald 9 metallic elements of Groups IA, IIA, [11A, 11B and VIII of Tmethod of 9 3 3 wherein sald sorbent materlal Is the Periodic Table, andmixed salts thereof to remove conmdal magneslum Sweatefree thyroxinetherefrom by selective sorption thereof by P0405) UNITED STATES PATENTOFFICE CERTIFICATE OF CORRECTIGN Patent'No. 3, 5" Dated y 3 972Inventor(s) Anna Eisentraut It is certified that error appears in theaboveidentified patent and that said Letters Patent are hereby correctedas shown below:

Col. 1, line 60 after "temperatures" delete "s"'.

Col. 2, line 36 "baRium" shouldbe -barium; Col. 2, line 42 after "($10delete ",ohl," and insert (OH) Col. 2, line 4" after (SiO delete ",ehand insert (OH) Col. 2, line 45 delete "Ialc" and insert --Talc-;

Col. 2, line 17 after (Si @0 (first occurrence) delete )()H)2" andinsert (OH) Col. 2, line 66 delete "l0" and insert lOO- 3 Col. 3, line63 delete "(OI-I and insert (OH) Col. 5, line 29 delete "b" Col. 6, line13 delete "globuline" and insert -globulin--; Col. 6, line 63 delete "33!l" and insert -33 .l- Col. 8, line 1 delete "combinations" and insert-combination--; Col. 8, line 2 delete "preferably" and insertpreferable- Col. 8, line 38 delete C" and insert 20C--; Col. 8, linedelete "7" (second occurrence) and insert 6--. Col. 9, line 3 delete"C." and insert --c-. Col. 10, line l delete "C. and insert -e Signedand sealed this 31st day of October 1972.

(SEAL) Attest:

EDWARD M.FLE TCHER,JR. v ROBERT GOTTSCHALK Attesting Office-rCommissioner of Patents L Q a

2. The improved test of claim 1 wherein the said particulate inorganiccrystalline sorbent material comprises colloidal magnesium silicate. 3.The improved test of claim 2 wherein said sorbing occurs at atemperature within the range of from about 20* C to about 30* C.
 4. Themethod of claim 3 wherein said sorbing occurs during a time interval ofat least about 2 minutes.
 5. The improved test of claim 4 wherein saidsample fluid comprises blood.
 6. A method for determining the level ofthyroid hormone in a sample of body fluid containing an unknown quantityof said hormone and hormone binding globulin comprising: a. thoroughlyadmixing a solution of this sample with a solution containing aradioactive isotope labeled hormone and barbital and allowing theresulting solution to equilibrate; b. thoroughly admixing the resultingequilibrated solution from step (a) with a particulate inorganiccrystalline sorbent material selected from silicic acid and carbonates,phosphates, oxides, hydroxides, silicates, aluminates, and sulfates ofthe metallic elements in Groups IA, IIA, IIIA, IIB, and VIII of thePeriodic Table, and mixed salts thereof to remove free thyroid hormonefrom said solution by selective sorption thereof by said sorbentmaterial; c. separating said sorbent from said solution; and d. countingwith a scintillation counter one of (1) the free radioisotope labeledhormone in said sorbent, and (2) the bound radioisotope labeled hormonein said resulting solution.
 7. The method of claim 7 wherein saidsorbent material is colloidal magnesium silicate.
 8. A method ofmeasuring thyroxine in a sample solution comprising: a. precipitatingproteins from said sample solution by admixing said sample solution withan organic solvent and thereby leaving said thyroxine in solution; b.thoroughly admixing a solution of the thyroxine from step (a) with asolution containing thyrobinding globulin, radioactive isotope labeledthyroxine, and barbital and allowing the resulting solution toequilibrate; C. thoroughly admixing the equilibrated solution of step(b) with a particulate inorganic crystalline sorbent material selectedfrom silicic acid and carbonates, phosphates, oxides, hydroxides,silicates, aluminates, and sulfates of the metallic elements of GroupsIA, IIA, IIIA, IIB and VIII of the Periodic Table, and mixed saltsthereof to remove free thyroxine therefrom by selective sorption thereofby said sorbent material; d. separating said sorbent material from theresulting solution; and C. counting with a scintillation counter one of(1) free labeled thyroxine removed in said sorbent, and (2) labeledthyroxine remaining in said solution.
 9. The method of claim 8 whereinsaid sorbent material is colloidal magnesium silicate.